There has been known a method for assaying endotoxin, which is a pyrogen, by using horseshoe crab amebocyte lysate (hereinafter referred to simply as lysate). This method has been employed as an official method as a substitute for the rabbit pyrogem test. This assay method is based on a reaction (hereinafter referred to as limulus reaction) of the coagulation of the lysate due to a trace amount of endotoxin. Subsequent biochemical studies have revealed that this reaction consists of stepwise activation of several coagulation factors (FIG. 1) [Takanori Nakamura, Nippon Saikingaku Zasshi (Japanese Journal of Bacteriology), 38, 781-803 (1983)]. The endotoxin serving as the trigger in this reaction is an outer membrane component of the cell wall of a gram negative bacterium and also called lipopolysaccharide (LPS). It is an amphiphathic substance having both of a hydrophilic sugar chain and a hydrophobic lipid A moiety in its molecule. Under normal conditions, subunits of the LPS form an extremely large molecular association of 10.sup.8 dalton via hydrophobic bonding, ionic bonding, etc. It is known that these subunits form a complicated micelle together with the proteins and lipids coexisting therewith. This micelle structure largely affects the reactivity of the LPS in the limulus reaction (hereinafter referred to as limulus activity) and there exist a molecular size attaining the maximum activity and the micelle structure thereof. When an anionic surfactant, such as cholic acid or deoxychollc acid, is added, the association is completely dissociated into monomers or dimers and thus the limulus activity is completely lost. An unstable micelIe not only causes changes in the assay data but also damages the stability of the endotoxin solution per se. In 1981, the Federal Food and Drug Administration (U.S.A.) attempted to improve an endotoxin reference standard originating in the E. coli 0113 strain by replacing the conventional additive albumin by lactose and polyethylene glycol to thereby stabilize the endotoxin activity. The reference standard thus obtained has been distributed by FDA as United States Pharmacopeia (USP) reference standard EC-5. In Japan, an attempt was made to employ mannitol as a filler of an endotoxin reference standard originating in E. coli UKT-B so as to stabilize the endotoxin activity by National Institute of Hygienic Science in 1988. The obtained product has been widely used as a reference standard of The Pharmacopoeia of Japan (JP).
However, USP and JP reference standards should be diluted prior to the assay, since they contain endotoxin at such a high concentration as 10,000 endotoxin unit (EU) per vial and 16,000 EU per vial, respectively. Thus dilution errors are unavoidable, which seriously hinders such an assay requiring a high accuracy and a high reproducibility as the quantitative limulus test. Namely, it is preferable to use an endotoxin reference standard which has been prepared in such a manner as to give a very low concentration. Thus, it has been required to develop a stable and highly reproducible additive therefor.
It is also known that when an endotoxin solution is dissolved in water and allowed to stand as such, the endotoxin activity of the aqueous solution is lowered within a short period of time, in particular, at a low concentration. Thus an endotoxin solution is generally prepared immediately before using and, after using, the residue should be discarded because of its poor stability, which not only requires a troublesome post-treatment but also causes economical disadvantage.
Accordingly, attempts have been made to store endotoxin in a lyophilized state to thereby solve the above-mentioned problems encountering in the case of the aqueous solution. However, there arises another problem, i.e., enlarged intervial variation. Namely, at a low endotoxin concentration, the content of endotoxin widely varies from vial to vial due to the scattering of the endotoxin powder.
There arises another problem that a lyophilized product cannot completely be dissolved upon use in some cases.
It is known in the field of medical services that routine control of endotoxin in body fluids is important in diagnosis of the symptoms of infectious diseases, in particular, sepsis, treatments of these diseases, judgement of the prognosis thereof, postoperative monitoring, etc. Also, it is essentially required in the safety management of dialyzates for patients under hemodialysis. In recent years, it is pointed out that the endotoxin activity in a dialyzate is unstable and lowered with the passage of time. It is therefore difficult to accurately determine endotoxin in a dialyzate, which seriously hinders the proper management of the dialyzate.